| Abstract: | Many tailed bacteriophages assemble ejection proteins and a portal–tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal–tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near–atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.Many double-stranded DNA (dsDNA) viruses, including tailed bacteriophages and herpesviruses, have a portal attached to a unique pentameric vertex of their icosahedral capsid shell (1–3). The portal is a dodecameric channel for viral DNA packaging and ejection. The tailed bacteriophages and herpesviruses encapsidate DNA in the capsid shell through the portal channel (4–10), and the last packaged DNA is held by tunnel loops (or β-hairpins for herpesviruses) in the portal (11–16). The last packaged DNA in most of the tailed bacteriophages and herpesvirus is the first to be ejected during the genome delivery (17). In tailed bacteriophages, the portal connects to a tail, which serves to recognize host cell receptors and deliver the genome into the cytoplasm (18). Gram-negative bacteriophage in Podoviridae initiate infection through a specific interaction of its receptor-binding protein with the receptor lipopolysaccharide (LPS) on the host cell surface. The phages in Podoviridae have a noncontractile tail that is too short to span the gram-negative bacteria envelope that comprises the outer membrane, the cytoplasmic membrane, and the peptidoglycan layer in the hydrophilic periplasm in between (19). After adsorption, a signal is transmitted for the release of internal ejection proteins to form a channel that extends the tail across the cell envelope and that allows for subsequent genome ejection into the infected cell (20–23). In many previous studies, structural analyses have been performed at resolutions of 9 to 40 Å on this highly coordinated dynamic infection process (21–26). These studies have provided insights on structural changes of phage particles that accompany the infection steps before and after the genome ejection. However, these studies did not resolve structures of the internal ejection proteins. Furthermore, the relative low resolutions cannot clarify the dynamic genome ejection process orchestrated by the ejection proteins, portal, and tail.Escherichia coli bacteriophage T7, a member of the Podoviridae family, has been used as a model for understanding the DNA packaging and delivery mechanism that are common to tailed phages and related dsDNA viruses (10, 21, 27–33). T7 has an icosahedral capsid shell formed by gene product 10 (gp10). The 12-fold portal (gp8) shares a very similar topology with those in other phages and herpesviurses (14–16, 30, 34). The tail comprises a 12-fold adaptor protein gp11 assembly, a sixfold nozzle protein gp12 assembly, and six subunits of trimeric tail fiber gp17 (21, 30). These tail fibers are responsible for bacterial receptor recognition and adsorption (21, 33). On top of the portal within the capsid shell is a hollow cylinder-shaped core structure (10, 28) formed by the ejection proteins (core proteins) gp14, gp15, and gp16, which have been suggested to form a transenvelope channel for the genome delivery into the infected cell (20, 35, 36). The gp16 harbors lytic transglycosylase (LTase) activity, which allows for penetration into the bacterial peptidoglycan layer (37).In this study, we present the structure of the mature bacteriophage T7 with internal core proteins at near-atomic resolution and the structures of the full and empty T7 particles in complex with their cell receptor at subnanometer and near-atomic resolutions, respectively. Our reconstruction reveals that the core in the mature T7 is formed by a fourfold gp16 ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The putative gp14 structures mediate the core–portal interaction. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a LTase domain. When the T7 phage interacts with the LPS, the tail nozzle opens. Six copies of gp14 anchor to the sixfold tail channel, extending the tail across the LPS lipid bilayer. A conformational change in the portal then triggers the genome ejection. Our structures reveal the structural changes of the phage genome-delivery molecular machines after the genome delivery. |
